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Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1.

Tibelius A, Marhold J, Zentgraf H, Heilig CE, Neitzel H, Ducommun B, Rauch A, Ho AD, Bartek J, Krämer A - J. Cell Biol. (2009)

Bottom Line: Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life.Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling.In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, 69120 Heidelberg, Germany.

ABSTRACT
Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

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MCPH1 recruits Chk1 to the centrosome via PCNT. (A) Centrosomal PCNT levels are reduced in MCPH1427insA and PCNT3109G>T LBCs relative to control lymphoblasts. Normal, MCPH1427insA, and PCNT3109G>T LBCs as well as U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) antibodies and analyzed by confocal microscopy. (B) Quantification of pixel intensity profiles constructed from optically sectioned (z axis) fluorescence images of normal cells. Error bars represent standard deviations from the analysis of 100 cells. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 7.2 × 10−6 (MCPH1427insA). (C) Control and Chk1−/− chicken DT40 cells as well as U2OS cells transfected with luciferase- or Chk1-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) and analyzed by confocal microscopy. (D) Endogenous PCNT and Chk1 interact with each other in vivo. Endogenous Chk1 was detected in immunoprecipitates using an anti-PCNT antibody in both control and MCPH1427insA LBCs. Reciprocally, endogenous PCNT was detectable after immunoprecipitation of Chk1 in both cell lines as well. Immunoprecipitation with an anti-HA antibody served as a negative control. Input represents 10% of the amount used for immunoprecipitation. White lines indicate that intervening lanes have been spliced out. (E) Cofractionation of PCNT, MCPH1, and Chk1 in U2OS whole cell lysates. Lysates were prepared from U2OS cells transiently transfected with GFP-MCPH1 24 h before lysis and size fractionated by fast protein liquid chromatography using a Superose 6 column. Proteins from consecutive fractions were analyzed by Western blotting using antibodies to PCNT, GFP, and Chk1. The size of marker proteins is shown on top. Numbers in red indicate the fractions that contain all three proteins (GFP-MCPH1, PCNT, and Chk1). Arrowheads point to centrosomes, which are shown enlarged in insets. Bars, 10 µm.
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fig3: MCPH1 recruits Chk1 to the centrosome via PCNT. (A) Centrosomal PCNT levels are reduced in MCPH1427insA and PCNT3109G>T LBCs relative to control lymphoblasts. Normal, MCPH1427insA, and PCNT3109G>T LBCs as well as U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) antibodies and analyzed by confocal microscopy. (B) Quantification of pixel intensity profiles constructed from optically sectioned (z axis) fluorescence images of normal cells. Error bars represent standard deviations from the analysis of 100 cells. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 7.2 × 10−6 (MCPH1427insA). (C) Control and Chk1−/− chicken DT40 cells as well as U2OS cells transfected with luciferase- or Chk1-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) and analyzed by confocal microscopy. (D) Endogenous PCNT and Chk1 interact with each other in vivo. Endogenous Chk1 was detected in immunoprecipitates using an anti-PCNT antibody in both control and MCPH1427insA LBCs. Reciprocally, endogenous PCNT was detectable after immunoprecipitation of Chk1 in both cell lines as well. Immunoprecipitation with an anti-HA antibody served as a negative control. Input represents 10% of the amount used for immunoprecipitation. White lines indicate that intervening lanes have been spliced out. (E) Cofractionation of PCNT, MCPH1, and Chk1 in U2OS whole cell lysates. Lysates were prepared from U2OS cells transiently transfected with GFP-MCPH1 24 h before lysis and size fractionated by fast protein liquid chromatography using a Superose 6 column. Proteins from consecutive fractions were analyzed by Western blotting using antibodies to PCNT, GFP, and Chk1. The size of marker proteins is shown on top. Numbers in red indicate the fractions that contain all three proteins (GFP-MCPH1, PCNT, and Chk1). Arrowheads point to centrosomes, which are shown enlarged in insets. Bars, 10 µm.

Mentions: As PCNT has an established role in targeting regulatory proteins to the centrosome (Diviani et al., 2000; Chen et al., 2004), direct or indirect binding of Chk1 by PCNT might mediate the centrosomal localization of Chk1 (Griffith et al., 2008). To determine whether the loss of Chk1 from centrosomes in MCPH1-deficient cells is a consequence of a shortage of centrosomal PCNT, control and MCPH1427insA LBCs were immunostained for PCNT. In contrast to control LBCs, centrosomal PCNT signals were diminished in MCPH1427insA cells (Fig. 3, A and B; and Fig. S2 A). Immunostaining of MCPH1 siRNA– and mock siRNA–treated U2OS cells with antibodies to PCNT led to similar results. Consistently, centrosome preparations from MCPH1427insA cells contained less PCNT as compared with control LBCs (Fig. 2 B). Also, GFP-MCPH1 coimmunoprecipitated with endogenous PCNT when transiently expressed in U2OS cells (Fig. S2 B). Similarly, immunoprecipitation of GFP-MCPH1 led to coimmunoprecipitation of endogenous PCNT. Thus, MCPH1 and PCNT interact with each other, and in addition to a loss of centrosomal Chk1, PCNT is depleted from centrosomes in MCPH1-deficient cells. To the contrary, PCNT deficiency did not impact on the abundance of MCPH1 at centrosomes in PCNT3109G>T or PCNT siRNA–treated U2OS cells (Fig. 2 A). Also, the centrosomal levels of PCNT remained unchanged in both U2OS cells transfected with Chk1 siRNA and Chk1−/− DT40 cells (Fig. 3 C; Zachos et al., 2003).


Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1.

Tibelius A, Marhold J, Zentgraf H, Heilig CE, Neitzel H, Ducommun B, Rauch A, Ho AD, Bartek J, Krämer A - J. Cell Biol. (2009)

MCPH1 recruits Chk1 to the centrosome via PCNT. (A) Centrosomal PCNT levels are reduced in MCPH1427insA and PCNT3109G>T LBCs relative to control lymphoblasts. Normal, MCPH1427insA, and PCNT3109G>T LBCs as well as U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) antibodies and analyzed by confocal microscopy. (B) Quantification of pixel intensity profiles constructed from optically sectioned (z axis) fluorescence images of normal cells. Error bars represent standard deviations from the analysis of 100 cells. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 7.2 × 10−6 (MCPH1427insA). (C) Control and Chk1−/− chicken DT40 cells as well as U2OS cells transfected with luciferase- or Chk1-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) and analyzed by confocal microscopy. (D) Endogenous PCNT and Chk1 interact with each other in vivo. Endogenous Chk1 was detected in immunoprecipitates using an anti-PCNT antibody in both control and MCPH1427insA LBCs. Reciprocally, endogenous PCNT was detectable after immunoprecipitation of Chk1 in both cell lines as well. Immunoprecipitation with an anti-HA antibody served as a negative control. Input represents 10% of the amount used for immunoprecipitation. White lines indicate that intervening lanes have been spliced out. (E) Cofractionation of PCNT, MCPH1, and Chk1 in U2OS whole cell lysates. Lysates were prepared from U2OS cells transiently transfected with GFP-MCPH1 24 h before lysis and size fractionated by fast protein liquid chromatography using a Superose 6 column. Proteins from consecutive fractions were analyzed by Western blotting using antibodies to PCNT, GFP, and Chk1. The size of marker proteins is shown on top. Numbers in red indicate the fractions that contain all three proteins (GFP-MCPH1, PCNT, and Chk1). Arrowheads point to centrosomes, which are shown enlarged in insets. Bars, 10 µm.
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Related In: Results  -  Collection

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Show All Figures
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fig3: MCPH1 recruits Chk1 to the centrosome via PCNT. (A) Centrosomal PCNT levels are reduced in MCPH1427insA and PCNT3109G>T LBCs relative to control lymphoblasts. Normal, MCPH1427insA, and PCNT3109G>T LBCs as well as U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) antibodies and analyzed by confocal microscopy. (B) Quantification of pixel intensity profiles constructed from optically sectioned (z axis) fluorescence images of normal cells. Error bars represent standard deviations from the analysis of 100 cells. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 7.2 × 10−6 (MCPH1427insA). (C) Control and Chk1−/− chicken DT40 cells as well as U2OS cells transfected with luciferase- or Chk1-specific siRNA were costained with rabbit anti-PCNT (green) and mouse anti–γ-tubulin (red) and analyzed by confocal microscopy. (D) Endogenous PCNT and Chk1 interact with each other in vivo. Endogenous Chk1 was detected in immunoprecipitates using an anti-PCNT antibody in both control and MCPH1427insA LBCs. Reciprocally, endogenous PCNT was detectable after immunoprecipitation of Chk1 in both cell lines as well. Immunoprecipitation with an anti-HA antibody served as a negative control. Input represents 10% of the amount used for immunoprecipitation. White lines indicate that intervening lanes have been spliced out. (E) Cofractionation of PCNT, MCPH1, and Chk1 in U2OS whole cell lysates. Lysates were prepared from U2OS cells transiently transfected with GFP-MCPH1 24 h before lysis and size fractionated by fast protein liquid chromatography using a Superose 6 column. Proteins from consecutive fractions were analyzed by Western blotting using antibodies to PCNT, GFP, and Chk1. The size of marker proteins is shown on top. Numbers in red indicate the fractions that contain all three proteins (GFP-MCPH1, PCNT, and Chk1). Arrowheads point to centrosomes, which are shown enlarged in insets. Bars, 10 µm.
Mentions: As PCNT has an established role in targeting regulatory proteins to the centrosome (Diviani et al., 2000; Chen et al., 2004), direct or indirect binding of Chk1 by PCNT might mediate the centrosomal localization of Chk1 (Griffith et al., 2008). To determine whether the loss of Chk1 from centrosomes in MCPH1-deficient cells is a consequence of a shortage of centrosomal PCNT, control and MCPH1427insA LBCs were immunostained for PCNT. In contrast to control LBCs, centrosomal PCNT signals were diminished in MCPH1427insA cells (Fig. 3, A and B; and Fig. S2 A). Immunostaining of MCPH1 siRNA– and mock siRNA–treated U2OS cells with antibodies to PCNT led to similar results. Consistently, centrosome preparations from MCPH1427insA cells contained less PCNT as compared with control LBCs (Fig. 2 B). Also, GFP-MCPH1 coimmunoprecipitated with endogenous PCNT when transiently expressed in U2OS cells (Fig. S2 B). Similarly, immunoprecipitation of GFP-MCPH1 led to coimmunoprecipitation of endogenous PCNT. Thus, MCPH1 and PCNT interact with each other, and in addition to a loss of centrosomal Chk1, PCNT is depleted from centrosomes in MCPH1-deficient cells. To the contrary, PCNT deficiency did not impact on the abundance of MCPH1 at centrosomes in PCNT3109G>T or PCNT siRNA–treated U2OS cells (Fig. 2 A). Also, the centrosomal levels of PCNT remained unchanged in both U2OS cells transfected with Chk1 siRNA and Chk1−/− DT40 cells (Fig. 3 C; Zachos et al., 2003).

Bottom Line: Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life.Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling.In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, 69120 Heidelberg, Germany.

ABSTRACT
Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

Show MeSH
Related in: MedlinePlus